The invention relates generally to the amplification of polynucleic acids, more particularly isothermal amplification of double-stranded DNA or RNA. More specifically, the invention relates to improvements to the loop-mediated isothermal amplification (LAMP) reaction, wherein minute amounts of DNA can be quantified using relatively inexpensive equipment without temperature cycling or the addition of successive reagents.
Various techniques exist that help speed up the amplification of nucleic acid to aid research. The polymerase chain reaction (PCR) is one of such techniques used to amplify DNA to generate many orders of magnitude copies of the sequence of interest. To initiate PCR, the two strands of the DNA are first separated into single strands during a denaturation step. This step is often achieved by breaking the hydrogen bonds between the two strands using high temperature (94-98° C.). Once the two strands are separated, temperature is lowered to between 50-60° C. so that primers may anneal to each of the single strands. Controlling the temperature is critical because the temperature must be low enough to allow hybridization between the primer and the strand to take place, but high enough for specificity so that hybridization will not occur unless the primer is perfectly complementary to the targeted sequence on the strand. DNA polymerase then binds to the primer-strand and begins DNA extension, creating a new strand with a sequence that is complementary to the single-strand serving as its template. Depending on the specific DNA polymerase used, the temperature during this extension step is typically increased to between 70-80° C. again to optimize the reaction.
This cycle of denaturation, annealing and extension are repeated until the desired order of magnitude of DNA fragments is made. With each cycle, the volume of DNA target is doubled, as each newly synthesized amplicon becomes another template after the denaturation step.
One drawback of PCR is the reliance on thermal cycling, which requires the use of precision cyclers to heat and cool the reaction to achieve the required temperatures of various steps of PCR.
Another technique for DNA amplification is loop-mediated isothermal amplification (LAMP). The LAMP method involves two specifically designed inner primers and two specifically designed outer (displacement) primers, targeting a total of six sequences, and a DNA polymerase with high strand displacement activity.
As shown in FIG. 1 (Notomi et al. Loop-mediated isothermal amplification of DNA, Nucleic Acids Research, (2000) Vol 28., No. 12), the sequences inside both ends of the target region for amplification are designated F2c and B2. Sequences outside the ends of F2c and B2 are designated F3c and B3, respectively; while the inner sequences from the ends of F2c and B2 are designated F1c and B1, respectively. In order to target the desired sequence for amplification, the forward inner primer (FIP) is specifically designed to include F1c and F2, which is complementary F2c. Similarly, the backward inner primer (BIP) is specifically designed to include B2 and B1c.
To start LAMP, targeted DNA and the four primers are heated to cause the double-stranded DNA to denature into single strands of DNA. The FIP hybridizes to F2c in the targeted single-stranded DNA and initiates DNA synthesis. Typically, primer F3 is added in lower concentration than FIP. F3 hybridizes to F3c (just outside of the F2c sequences) and begins DNA synthesis and displaces the FIP linked complementary strand that was initiated by the hybridization of F2c to F2. This displaced single strand forms a loop at one end, hybridizing F1c of the FIP to F1 region of the synthesized amplicon. This single loop amplicon serves as the template for BIP-initiated DNA amplification. Primer B3 then hybridizes and initiates strand displacement DNA synthesis, releasing the BIP-linked complementary strand created, leading to the production of a dumbbell form amplicon having two loops, one at each end of the amplicon. This dumbbell form amplicon structure is quickly converted to a stem-loop amplicon by self-primed DNA synthesis (structure 7). This stem-loop amplicon then serves as the starting material for LAMP cycling.
To initiate LAMP cycling, FIP anneals to the single-stranded region in the loop in the stem-loop amplicon (structure 7) and primes strand displacement DNA synthesis, releasing the previously synthesized strand. An intermediate one gapped stem-loop DNA with an additional inverted copy of the target sequence in the stem and a loop formed at the opposite end via the BIP sequence (structure 8) are produced. Subsequent self-primed strand displacement DNA synthesis yields one complementary structure of the original stem-loop amplicon (structure 10) and one gap repaired stem-loop amplicon with a stem elongated to twice as long (double copies of the target sequence) and a loop at the opposite end (structure 9). Both of these products then serve as template for a BIP-primed strand displacement reaction in the subsequent cycles. The final products are a mixture of stem-loop amplicons with various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand.
To accelerate LAMP, loop primers may be optionally used. The loop primer is designed to anneal to binding sites in the single-stranded loop region, either between B1 and B2 regions or between F1 and F2 regions on the amplicons. Without loop primers, LAMP cycling occurs with FIP and BIP initiating DNA synthesis only at sites F2c or B2c. With the addition of loop primers, loops including F2 and B2 sites are also used for further DNA synthesis, thereby increasing the speed and efficiency of amplification.
LAMP may also be accelerated by the use of stem primers. Stem primers target the stem portion of the stem-loop amplicon and do not bind to the single-stranded DNA loops. Stem primers are designed to target specific sequences between B1 and F1c, and F1 and B1c. These areas are theorized to be transiently single-stranded during early stages of amplicon formation, and thus stem primers have been theorized to be helpful in speeding up LAMP reactions since they target single-stranded regions to form new amplicons. Multiple stem primer sets may be designed for longer stems.
The mechanism of stem-accelerated LAMP is similar to LAMP in that the annealing and extension of FIP/BIP primer cause the displacement and release of the opposite strand. This released single strand provides a binding site for the stem primer even before the loops are formed to provide binding sites for loop primers. The stem primer hybridizes to the single strand and initiates DNA synthesis and the displacement of the FIP/BIP-initiated amplicon, producing another amplicon that may serve as a template. Theoretically, both stem primers and loop primers may be used in a single amplification. In reality, however, it may be difficult to find ten binding sites available on the targeted sequence.
Unlike PCR, the reactions of the steps involved in LAMP and stem-accelerated LAMP are carried out in reaction mixtures maintained at a constant temperature. Stem-accelerated LAMP may have advantages over LAMP, as stem primers offer more flexibility and less restriction. Stem primers also do not require to be designed with a specific orientation, while LAMP primers do. Stem-accelerated LAMP may also be carried out without displacement primers, which may be especially useful if the targeted sequence is short and cannot accommodate all eight binding sites.
Whereas LAMP provides a significant advantage over other methods of DNA amplification, it has drawbacks as well. LAMP generally requires pre-amplification heat or chemical denaturation, and has moderate reaction variability, which increases with lower template concentration reactions. LAMP also has moderate reaction speed, and whereas the results are generally detectable by the naked eye, LAMP provides relatively weak color/turbidity changes. Furthermore, since the primer system is complex, conventionally requiring four to six primers targeting six to eight distinct regions in the target DNA, it may be difficult to design high-performance primer sets to amplify a desired target sequence.
Accordingly, it is desirable to provide an improved system and method for DNA amplification that overcomes drawbacks and inadequacies of known methods and systems.